Project Summary: Bacterial cell surface glycolipids function in critical roles in the adaption of bacteria to their environment and pathogenesis. Owing to their extracellular location and functional importance, considerable work has been devoted to delineating bacterial glycolipid biogenesis (biosynthesis and transport). While significant advances have been made in the identification of transporters in particular in Gram-negative bacteria, these processes remain wholly opaque in the specialized Gram-positive organisms belonging to the order Actinomycetales . Actinomycetales of the mycolata group (i.e., mycolic acid containing species), which contain notorious human pathogens, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, are characterized by a cell envelope of unique composition and structure suggestive of highly specialized biosynthetic and translocation machineries. Some of the most studied glycans in the cell envelope of mycolata species for the important roles they play in physiology and pathogenesis are mannosylated glycolipids and lipoglycans known as phosphatidylinositol mannosides (PIMs) and their multi-glycosylated counterparts, lipomannan (LM) and lipoarabinomannan (LAM). While many enzymes in the PIM, LM, and LAM biosynthetic pathway have been discovered, the transporter(s) responsible for translocation of PIM intermediates across the plasma membrane, and of PIM, LM and LAM to the outer membrane (mycomembrane) and cell surface are not known. This application proposes to gain the first insights into the molecular mechanisms governing PIM, LM and LAM translocation across the different layers of the cell envelope of mycolata group bacteria. A current obstacle to the discovery of these elusive transporters is the lack of biochemical tools to topologically and specifically label PIMs. To address this deficiency, I have devised a multidisciplinary approach encompassing chemical biology, glycobiology, genetics and protein-protein interactions. First, I will develop a set of tools that can directly label PIMs that have undergone translocation across the plasma membrane [Aim 1]. These tools, in combination with traditional genetic, biochemical and protein-protein interaction approaches, and a newly developed cell sorting- based assay to screen of a transposon mutant library will enable the identification of specific PIM and LAM transporters [Aim 2]. Success in these studies would advance long-standing questions regarding (glyco)lipid transport in Actinomycetes, and PIM, LM and LAM biogenesis in mycobacteria in particular. The new knowledge generated therein and translocation assays arising from Aim 1 may further find applications in the development of innovative therapeutic strategies to treat Corynebacterineae infections.